Studies of the interaction of proteins or DNA with small ligands are commonplace in biochemical, pharmacologic, and toxicological research. To detect the interaction of macromolecules with their specific ligands in solution, one can either measure binding-induced changes in physical properties of the macromolecule or of the ligand (optical, NMR, etc.) or use some partitioning technique (equilibrium dialysis, size-exclusion chromatography, etc.). Each of these approaches has serious limitations. For example, the optical properties of a macromolecule will change only if some reporter group (fluorescent or absorbent) is by chance situated near the binding site. On the other hand, partitioning methods require the determination of small concentrations of ligands, which often necessitates synthetic attachment of radioactive or chromogenic labels. This makes the procedure expensive, tedious, and time consuming. Some of these negative features have been avoided recently by the use of titration calorimetry. Nevertheless, in research on a large series of ligands, considerable consumption of protein and DNA in solution may be a disadvantage to all these methods.
To explain further: Assays of protein specificity can also be classified according to how they are performed-either in solution (homogenous assays) or in a heterogeneous system, in which the protein or specific ligand is immobilized (heterogeneous assays). The performance characteristics, advantages and disadvantages of all these methods, are determined primarily by the detection technique used.
In homogeneous methods, binding of a ligand to a protein is detected by a physical (mostly optical) method. With few exceptions, homogeneous methods are not universal, since only binding accompanied by large conformational changes in proteins or binding with sites having a neighboring reporter group can be reliably detected. Microcalorimetry and equilibrium dialysis are examples of more general homogeneous methods. However, the former requires relatively large quantities of proteins for each assay; since protein samples are not reusable, screening by this method is material consuming. On the other hand, equilibrium dialysis is slow and requires sensitive methods for determining ligand concentrations if small amounts of protein are used.
Heterogeneous methods are more economical, in that they require much less protein for each sample and the protein sample can be reused. Binding in heterogeneous systems can be detected either directly by changes in physical properties of a protein layer (mass, optical properties, etc.), or indirectly, by competitive replacement of a labeled ligand by a substance under study. Direct methods can identify binding to any site in a protein, whereas indirect methods detect binding at known sites, overlooking ligand binding at secondary sites. This is why indirect heterogeneous methods cannot be used for primary screening of a protein with unknown function or binding site.
Heterogeneous methods encounter serious difficulties in analysis of binding of small ligands (Karlsson, R., Analyt. Biochem., 221:142-151 (1994)). For direct methods, this is due to the fact that small changes in mass and in optical properties of protein layer accompanying binding of small ligands are involved. In indirect methods, labeling of small ligands themselves presents a problem, since introduction of a fluorophore or enzyme label might alter the specificity of the labeled ligand. Radioactive labeling is free of this drawback, although it is considered to be dangerous and expensive, since radioactive ligands for each protein must be available.
The present inventors originated a mechanochemical method of using the mechanical elastic characteristics of a protein film to measure ligand-protein interaction. The method is described in two publications: V. N. Morozov and T. Ya. Morozova (1992) "Mechanical Detection of Interaction of Small Specific Ligands with Proteins and DNA in Cross-Linked Samples", Analytical Biochem., 201:68-79; and in V. N. Morozov and T. Ya. Morozova (1984) "Protein Molecule as a Bioanalytical Device", FEBS Letters, 175:299-302, the entire contents of both of which are hereby incorporated herein by reference.
In those articles, the inventors disclosed a new method for detecting the interaction of macromolecules with their specific ligands by exploiting the ability of cross-linked protein and DNA solid and gel samples to change their mechanical properties upon binding of the specific ligands with the macromolecules. This mechanochemical phenomenon was initially discovered in lysozyme crystals (Morozov et al, J. Mol. Biol., 157:173-179 (1982), Morozov et al, Biophysics (Sov.), 28:786-793 (1983)) and in papain films (Morozov et al, FEBS Lett., 175:299-302 (1984)). The inventors suggested that the phenomenon has some universal character because it is based on two well-known facts. First, many crystalline proteins retain their ability to bind specific ligands. This fact is widely used in the X-ray analysis of protein-ligand complexes (Rupley in Structure and Stability of Biological Macromolecules, Timasheff and Fasman, Eds., pp. 291-353, Dekker, New York (1969)). Second, whatever the character of this binding, to some extent it changes inter- and intramolecular interactions, the molecular structure, and the packing of molecules in the solid sample.
Of all the physical parameters of protein solids, the mechanical ones seem to be the most sensitive to these changes. Theoretical analysis shows that deformation of protein molecules by 10.sup.-6 .ANG. can be, in principle, measured with mechanical methods. In practice, changes in average protein dimensions of 10.sup.-2 .ANG. can be readily detected.
In order to prepare samples for mechanochemical testing, the inventors used a process shown in FIG. 1. A salt-free solution (10-200 mg/ml of protein) was poured onto a glass plate and rapidly dried under a reduced atmospheric pressure of 10-15 mm Hg. The resulting dry protein film (5-10 .mu.m thick) was then cross-linked in a vapor of 25% glutaraldehyde (GA) solution at 25-27.degree. C. The time required to obtain insoluble film varied between 0.5 and 6 h for different proteins. The cross-linked film was washed with water, to remove the excess aldehyde, and then carefully detached from the glass plate.
DNA films were prepared by dialyzing a DNA precipitate in alcohol against a 10 mM NaCl solution to give a gel with a DNA concentration of about 100 mg/ml. The gel was then distributed over the glass surface, dried as described for protein films, and cross-linked by UV irradiation under a 30 W germicidal lamp located 0.1 m from the sample for 1.5 h (Sheldon et al, Proc. Natl. Acad. Sci. USA, 62:417-421 (1972)).
Strips 300-700 .mu.m long and 20-50 .mu.m wide were cut with a razor blade to be used as samples. These samples were then mechanically tested while immersed in a ligand solution. The mechanical testing involved stretching the strip lengthwise, letting it relax under isometric condition and then measuring changes in isometric tension in response to immersing the sample into ligand solutions.
In their prototype method, the inventors employed an apparatus that is shown in FIG. 2, a partly schematic view labeled "prior art". The apparatus uses two devices to stretch the sample. One is a bimorph type transducer, used mostly to periodically stretch the sample when measuring its elastic modulus. Because practical deformation of piezomaterials under dc voltage is not large enough and not stable enough, another device is used to apply constant strain and to keep it precisely isometric during testing. This is done by a rotating quartz plate (105 in FIG. 5; not shown in FIG. 2). Thus, the apparatus uses a rotating quartz plate and piezo-electric bimorph to stretch the prepared strip in static and dynamic ways respectively and a variable-capacitance transducer to measure the static and dynamic stresses in the sample in response to the deformations and ligand applications.
Connected to a bedplate or base 110 are a flow chamber 180, strain transducer 140, and force transducer 160. The flow chamber 180 includes a well 181 full of ligand solution, which is held in the well by gravity and surface tension. The solution in the open top of the well 181 is bounded by a meniscus M. The solution is fed into the well 181 through a pipe 182 and is sucked off the top of the meniscus M by a suction tube 183. Inside the meniscus M and the sides of the well 181 is a sample strip S held by pincers 168 and 148, which are attached to their respective transducers. Flow chamber 180 is mounted on the device housing and is capable of moving up and down, thus enabling dipping the pincers into chamber (in up position) and enabling an easy access to the pincers (on sample attachment) in down position. The pincers 168 and 148 pass through L-shaped slots in opposite sides of the well 181, so that the pincers are free to move over restricted ranges before they contact the sides of the well 181. The strip mounting and pincers 168, 148 are described more fully below in regard to FIG. 3.
Still considering FIG. 2, the strain transducer 140 includes a rectangular member mounted on the base 110 by a mounting bracket 146. This rectangular member is a piezoelectric bimorph, that is, a sandwich of two layers of piezoelectric material 142 and 143 which are oriented at 180 degrees to one another in such a way that, when an electric field is applied across the sandwich 142/143, one of the layers contracts along the member's length and the other layer expands. As in a bi-metallic thermostat, when one layer expands and the other contracts the member as a whole bends. To apply the electric field needed to bend the sandwich 142/143, both sides of the sandwich member are coated with metal. A metal layer 141 is coated on the underside of piezo layer 142, and a metal layer 144 is coated on the top side of piezo layer 143. Leads 2142 and 2143 connect the metal layers 142 and 144 respectively to an adjustable voltage source (not shown in FIG. 2). By adjusting the applied voltage, the pincer 148 can be moved a calibrated amount.
FIG. 3, also labeled "prior art", shows how the pincers 168 and 148 are connected within the well 181. In each of the pincers 168, 148 the strip S is held by pliers-like pincer jaws formed by splitting a tungsten wire; the split wires are tightened onto the sample S by a sliding ring 146.
The pincer 168 is firmly attached to a resilient cantilevered beam 162, which is mounted on the base plate 110 by a bracket 166. When the strip S in the well 181 is moved by the transducer 140, the beam 162 is deflected by a certain amount; due to this deflection, the beam 162 exerts a restoring force that is a function of its deflection. Therefore, by measuring the deflection of the beam 162 the force or stress in the sample S can be measured. To measure the beam deflection a capacitor is formed as follows: the underside of the beam 162 is coated with a first layer of metal 164 and a block 169, mounted on the base 110, is covered with a second layer of metal 161.
The metal layers 161 and 164 form a parallel-plate capacitor whose capacitance varies with the gap between the metal layers 161, 164; that is, with the deflection of the beam 162, which deflection is proportional to the force in the strip S, within a fraction of a percent. The metal layers 161, 164 are connected by leads 2161, 2164 to circuitry (not shown) which measures the capacitance of the layers electronically (for example, by measuring the frequency of an LC resonant circuit which includes the beam 162 capacitor as the C value and a remote inductor as the L value). The measured capacitance is thus a measure of the tensile force along the strip S.
It is to be noted that the displacement of the pincer 148 is equal to the sum of the displacement of the pincer 168 plus the elongation of the strip S.
The inventors' prototype method was a substantial advance in the art, being a new method of measuring ligand attachment that allowed new sorts of data to be collected. However, the prototype method of sample preparation and the force/strain measuring apparatus used had various drawbacks.
One drawback was that the very small and fragile samples, only 0.3-0.7 mm long, were difficult to handle and clamp for the stress/strain measurements.
The clamping arrangement shown in FIGS. 2 and 3 was quite awkward to use at the sub-millimeter scale. The flimsy strip S needed to be manipulated with great care. The preparation steps involving scraping and cutting the strips needed to be performed under a stereoscopic microscope, and placing the strips S into the pincer jaws required a micromanipulator. The procedure was very time-consuming.
The prototype apparatus of FIG. 3 is likely to grip the sample unevenly: that is, the jaws are quite likely to grip the sample harder at one point along their length than elsewhere, leading to uneven strain across the width of the sample S when the less tightly-gripped portion slides out. They are also likely to grip the sample at different points along the length, so that the effective length of the sample can be indeterminate or even uneven, in spite of the sample S being gripped with equal force at all points across the width. The pincer jaws may even tear the sample S.
The effective or gripped length of the strip S is one of the quantities that is used to calculate the quantitative data that the inventors' method yields. The quantitative results thus could easily be affected by uneven gripping. (The theory of the method is described below in the Detailed Description of the Invention.)
The bad effects of uneven sample gripping could be eliminated by high-precision jaws, but these would be very expensive and easily damaged; and if they were damaged, the damage would not be apparent.
An additional drawback of the prototype mechanical arrangement is that the offset of the two sets of pincer jaws cause fluid-flow problems. The jaws' vertical insertion movement and horizontal test motions require L-shaped slots in the flow chamber, and a flow chamber with such slots is not able to handle liquids with low surface tension, such as organic solvent solutions or water solutions containing surface active compounds, because these would simply leak out; only liquids which do not "wet" the well 181, and therefore will not run out of the slots, can be used.
Another drawback of the FIG. 2 apparatus is that the capacitive force transducer 160 is ill-protected from moisture, dust, and stray electric fields which can affect its readings.